Download

Download Full Text (256 KB)

Faculty Advisors

Thomas Gerken - Case Western Reserve University - Department of Biochemistry

Description

Mucin type O-glycosylation is a common modification of proteins in higher organisms found on cell surfaces and secreted proteins. O-glycosylation is important in a variety of biological processes from development, the control of serum phosphate levels, and playing role in vascular and heart diseases and cancer. A family of 20 transferases (GalNAc-Ts) initiate mucin type O-glycosylation in humans by adding the sugar GalNAc onto Ser or Thr residues on targeted proteins. Since several diseases and cancers are specifically linked to the expression of individual GalNAc-T isoforms, there is a need to fully understand and characterize their specificities and determine what their protein targets are. The GalNAc-Ts contain a lectin domain and a catalytic domain that our lab has shown are involved in what their protein targets are. Therefore, each GalNAc-T has its own unique combination of specificities that recognizes peptide sequence and prior remote and neighboring GalNAc substrate glycosylation. There is a poor understanding of what sites glycosylated in vivo tissues and what transferase isoform(s) performs the glycosylation. This is difficult due to the heterogeneous nature of the O-linked glycans due to glycan chain elongation. In addition, the elongated glycans interfere with nearby protease sites, making it difficult to isolate O-linked glycopeptides. Together, these aspects make it difficult to obtain it and analyze sites of O-glycosylation in tissues. The Gerken Lab has developed a chemical approach using mild trifluoromethanesulfonic acid (TFMSA) that can trim the extended O-linked glycans to the GalNAc residue. This approach eliminates glycan heterogeneity, permits better protease cleavage by trypsin and allows efficient glycopeptide isolation by lectin chromatography and in addition, greater ease of mass spectrometry (MS) analysis. My work was involved in helping to optimize the TFMSA, Trypsinolysis and lectin isolation steps. This involved performing lectin based Western blotting of the TFMSA reaction time course, demonstrating the inactivation of trypsin after sample digestion and to demonstrate the GalNAc-glycopeptide binding capacity of the lectin columns. My work on the TFMSA reaction time course gave inconclusive results thus more work needs to be done here. My study of the trypsin inactivation by heat was successful and my binding studies of the lectin column demonstrated that one of the columns had been partially inactivated. In addition, I began the processing of heart and muscle tissues from mice. Overall, these studies will be useful for the development of analytical approaches for determining sites of O-glycosylation in multiple tissues and will eventually be very useful in understanding what steps are O-glycosylated and which GalNAc-T isoforms are involved.

Publication Date

2019

College

College of Sciences and Health Professions

Student Publication

This item is part of the McNair Scholars Program.

Optimization of Isolation of O-Linked Glycopeptides

Share

COinS